Structure and function of the E. coli ribosome will be studied, with particular emphasis on how structural changes affect response to certain antibiotics (kasugamycin, erythromycin). Resistance to Ksg is the result of undermethylation of 16S RNA. We will study binding of labeled drug to normal and KsgR 16S RNA, as well as to complexes of 16S RNA and certain 30S proteins. Ksg acts to inhibit initiation of protein synthesis, but only inhibits initiation of certain phage-specific proteins; we will study whether this is due to specificity of interaction with certain initiation factors, or other reasons. We will also study the physiological role of methylation of 16S RNA; preliminary experiments indicate that undermethylated (KsgR) 16S RNA results in significant restriction of translational ambiguities, but has no effect on ribosome assembly. We have found a gene (ramB) near ksgA which results in alteration of 30S protein S4; this is of particular interest because the putative structural gene (ramA) for 30S protein S4 is located considerably distant from ramB, in the ribosomal gene cluster around str. We will study whether ramB is the locus for an enzyme which modifies S4 (phosphorylation or similar change). If so, this raises the possibility of a gene cluster of enzymes near ksgA which modify ribosome structure; we will search for other similar enzymes. We also will analyze the structure of several ribosomal proteins which are of special interest, by two-dimensional chromatography of tryptic digests, and sequence analysis of unusual peptides. Included will be normal 30S protein S4, and altered S4 resulting from mutations near str (ramA) and near ksgA (ramB), as well as the single (50S) protein which is altered in our Ery-dependent mutant and its high- frequency revertants to independence.